Use of intratracheal administration of SOD to protect humans from lung injury due to hyperoxia and hyperventilation

ABSTRACT

The subject invention provides a method of protecting a human from lung injury due to hyperoxia and hyperventilation which comprises intratracheally administering to the human an amount of free CuZnSOD effective to protect the human from lung injury due to hyperoxia and hyperventilation.

BACKGROUND OF THE INVENTION

Throughout this specification, various publications are referenced byArabic numerals within parentheses Full citations for these referencesmay be found at the end of the specification immediately preceding theclaims. The disclosures of these publications in their entireties arehereby incorporated by reference in this specification in order to morefully describe the state of the art to which this invention pertains.

Newborn infants with a variety of respiratory disorders are treated withpositive pressure mechanical ventilation and oxygen therapy Thistreatment is therapeutic, but can initiate a potent inflammatoryresponse leading to acute lung injury and subsequent BronchopulmonaryDysplasia or BPD. BPD begins as an acute lung injury that initiates aseries of inflammatory responses which then evolve into chronic lungdisease The exact mechanisms responsible for pathophysiologic disruptionto the lung in infants with BPD are not completely understood Onepossibility is that oxidative insult caused by superoxide radicals isresponsible for the initial, acute lung injury which ultimately leads tothe development of BPD. If acute lung injury could be ameliorated, thenit may be possible to prevent BPD.

Superoxide is a highly toxic free radical that may be an importantcomponent of pulmonary oxygen toxicity (1). SOD facilitates theconversion of superoxide radicals (O₂ ⁻) to , hydrogen peroxide (H₂ O₂).However Crapo et al. found that free superoxide dismutase, administeredby intraperitoneal injection or by aerosolization, failed to modifyeither the time course or the cumulative toxicity of 100% oxygen inadult rats (1).

Other experiments have been performed using free and liposome entrappedsuperoxide dismutase to determine the effect on pulmonary oxygentoxicity in rats (2,3). Seo et al. observed the effect ofintratracheally administered free and liposome entrapped bovinesuperoxide dismutase and catalase by measuring the enzyme activity,oxygen radicals, pulmonary hemorrhage and survival rate after exposureto hyperoxia in adult rats (2) Free superoxide dismutase delivered toadult rats intratracheally with catalase had no protective effectagainst hyperoxia lung damage, while the intratracheally-administeredliposome-entrapped superoxide dismutase and catalase enhanced theactivity of total superoxide dismutase, Mn-superoxide dismutase andcatalase in the rats (2). Padmanabhan et al. found that priorintratracheal administration of liposome-encapsulated bovineCuZn-superoxide dismutase produced a significant increase in theactivities of these enzymes in the lung tissue of rats exposed tohyperoxic levels of oxygen compared with that in control animals underthe same conditions with free superoxide dismutase (3).

As demonstrated by the references discussed above (1-3), until now,attempts to protect animals from lethal effects of pure oxygen byintratracheal administration of free superoxide dismutase have beenunsuccessful. The subject invention provides a method forintratracheally administering free human CuZn superoxide dismutase toprotect humans from lung damage due to hyperoxia and hyperventilation.

SUMMARY OF THE INVENTION

The subject invention provides a method of protecting a human from lunginjury due to hyperoxia and hyperventilation which comprisesintratracheally administering to the human an amount of free CuZnSODeffective to protect the human from lung injury due to hyperoxia andhyperventilation.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Percent polymorphonuclear (PMN) chemotactic activity in theneonatal piglet of group I (hyperventilated and hyperoxygenatedpiglets); group II (hyperventilated and hyperoxygenated piglets given asingle intratracheal dose of superoxide dismutase at time zero); andgroup III (normally ventilated piglets, under normoxia conditions,negative control group).

FIGS. 2A and 2B: Tracheal aspirate cell counts and number of PMNs in theneonatal piglet of group I (hyperventilated and hyperoxygenatedpiglets); group II (hyperventilated and hyperoxygenated piglets given asingle intratracheal dose of superoxide dismutase at time zero); andgroup III (normally ventilated piglets, under normoxia conditions,negative control group).

FIGS. 3A and 3B: Elastase activity and albumin in the neonatal piglet ofgroup I (hyperventilated and hyperoxygenated piglets); group II(hyperventilated and hyperoxygenated piglets given a singleintratracheal dose of superoxide dismutase at time zero); and group III(normally ventilated piglets, under normoxia conditions, negativecontrol group).

FIG. 4: Lung compliance per kg in the neonatal piglet of group I(hyperventilated and hyperoxygenated piglets); group II (hyperventilatedand hyperoxygenated piglets given a single intratracheal dose ofsuperoxide dismutase at time zero); and group III (normally ventilatedpiglets, under normoxia conditions, negative control group).

FIG. 5: Concentration of superoxide dismutase in the serum of theneonatal piglet of group I (hyperventilated and hyperoxygenatedpiglets); group II (hyperventilated and hyperoxygenated piglets given asingle intratracheal dose of superoxide dismutase at time zero); andgroup III (normally ventilated piglets, under normoxia conditions,negative control group).

DETAILED DESCRIPTION OF THE INVENTION

The subject invention provides a method of protecting a human from lunginjury, particularly lung injury due to hyperoxia and hyperventilation,which comprises intratracheally administering to the human an amount offree CuZnSOD effective to protect the human from such lung injury.

In one embodiment of the method, the human is an adult; in another, thehuman is a neonate; and in a further embodiment, the neonate ispremature. In practicing the method of this invention, a surfactant maydesirably be administered prior to administering the free CuZnSOD to thepremature neonate.

As used herein "CuZnSOD" means a polypeptide having an amino acidsequence identicial, or substantially identical, to the amino acidsequence of a naturally-occurring CuZnSOD, whether of human, bovine orother mammalian origin, and having the biological activity of suchnaturally-occurring CuZnSOD. Thus, CuZnSOD encompasses polypeptideswhose amino acid sequence varies from that of the naturally occurringCuZnSOD by one or more amino acid, either internally such as a pointmutation, or by addition or deletion at the COOH⁻ terminus or NH₂ ⁻terminus or both, provided that the biological activity is unchanged.

In the presently preferred embodiment of the invention the CuZnSOD is anonacetylated, nonglycosylated polypeptide analog of human CuZnSODprepared in E. coli (see U.S. Pat. No. 4,742,004, the disclosure ofwhich is hereby incorporated by reference into the present application).As used herein, "free CuZnSOD" means CuZnSOD which is neitherencapsulated nor bound to another polymer such as polyethylene glycol(PEG) or another polypeptide such as albumin.

The amount of CuZnSOD intratracheally administered may vary from about0.5 mg/kg to about 50 mg/kg of body weight of the human being treated.Preferably, the amount is from about 5 mg/kg to about 10 mg/kg, forexample about 5 mg/kg body weight of the human being treated. Theintratracheal free CuZnSOD may be administered in any other of themethods well known to those skilled in the art. For example, the CuZnSODmay be administered in the form of an aerosol or may be administered byinstillation. If administered in the form of an aerosol, a nebulizer isused to produce CuZnSOD in aerosol form.

Typically, the free CuZnSOD is administered in a pharmaceuticallyacceptable carrier, for example a saline solution. Such carriers arewell known in the art and the specific carriers employed may be varieddepending upon factors such as size of the subject being treated,treatment dose and the like.

Further, the time over which the CuZnSOD is administered may vary as iswell known in the art to achieve the desired results, for example, thefree CuZnSOD may be administered as an aerosol for from about 30 minutesto about 3 hours per treatment regimen (e.g. one daily).

In addition, forms of SOD other than CuZnSOD may be substituted forCuZnSOD in the method of this invention, such as MnSOD, extracellularSOD or analogs thereof. If MnSOD is substituted for CuZnSOD, thepreferred amount of MnSOD intratracheally administered to the human isfrom about 0.1 mg/kg to about 50 mg/kg of body weight of the human.

As used herein "MnSOD" means a polypeptide having an amino acid sequenceidenticial, or substantially identical, to the amino acid sequence of anaturally-occurring MnSOD, whether of human, bovine or other mammalianorigin, and having the biological activity of such naturally-occurringMnSOD. Thus, MnSOD encompasses polypeptides whose amino acid sequencevaries from that of the naturally occurring MnSOD by one or more aminoacid, either internally such as a point mutation, or by addition ordeletion at the COOH⁻ terminus or NH₂ ⁻ terminus or both, provided thatthe biological activity is unchanged.

EXPERIMENTAL DETAILS

The subject invention involves the acute response of the lung to injurycaused by oxygen and mechanical ventilation and demonstrates thatprophylactic superoxide dismutase (SOD) could prevent this injury.

A multidisciplinary approach was used to study the biochemical,cellular, biophysical, physiologic, and pathologic effects of oxygen andmechanical ventilation on the newborn lung. The neonatal piglet has beenchosen as the model for these experiments. The piglet is large enough atterm for ventilation and for the study of physiologic variables. Thelung of the newborn piglet is similar physiologically andmorphologically to that of a preterm human infant. Anti-oxidant enzymeactivity of the newborn piglet is also comparable to that of a pretermhuman infant.

The piglet model of the subject invention is a better model than thepreviously used rat model for humans for these experiments because rats,unlike humans, can be induced to survive in a 100% O₂ environment(2,4,5). In Walther et al. (6), the first experiment performed usingpremature animals instead of rats, polyethylene glycol (PEG) superoxidedismutase was delivered with PEG-catalase intravenously to prematurelambs, which resulted in decreased lung damage during subsequentmechanical ventilation (6). However, although lambs are a better modelfor humans than are rats, the subject invention differs from theexperiment with premature lambs because the superoxide dismutase used inthe subject invention is free CuZn superoxide dismutase, not derivatizedPEG-SOD, is delivered alone, not in combination with PEG-CAT, and isdelivered intratracheally, not intravenously, to neonatal piglets. Innewborn piglets, 48 hours of hyperoxia and hyperventilation causes acutelung injury as evidenced by biochemical, cellular and pathophysiologicchanges in the lung (7). This experiment demonstrates that prophylacticsuperoxide dismutase (SOD) prevents such lung injury to newborn piglets.

To determine if prophylactic SOD would prevent acute lung injury causedby hyperoxia and hyperventilation, 24 piglets (1.3±0.3 kg, 1-2 days old)were studied. Ten piglets were hyperventilated (PaCO₂ 15-20 torr) with100% O₂ 2 for 48 h (group I, pos. control) and compared to 8 identicallytreated piglets given a single intratracheal dose of SOD (5 mg/kg insaline) at time zero (group II). Six piglets were normally ventilated(PaCO₂ 40-45 torr) with 21% O₂ (group III, neg. control). Pulmonaryfunction and tracheal aspirates were examined at 0, 24, and 48 h.Morphologic and surfactant(s) analyses were performed at the conclusionof the study. In group I, there was a significant decrease in lungcompliance and an increase in tracheal aspirate cell counts, number ofneutrophils (PMNs), PMN chemotactic activity, elastase activity andalbumin and total protein concentrations over 48 h. Morphologicexamination revealed patchy, moderate atelectasis, edema, inflammationand cell necrosis. In contrast the only significant change in group IIwas aspirate total protein concentration. Changes in cellular,biochemical and lung morphologic variables in group II weresignificantly less than group I and comparable to group III controls.Serum SOD levels peaked at 0.6±0.2 μg/ml at 12 h and were 0.2±0.05 μg/mlat 48 h. Lung tissue SOD was 8.5±3.6 μg SOD/mg protein at 48 h. S wasactive in all groups. When SOD was added directly to tracheal aspiratesfrom group I (0.5, 5, 50 μg/ml), no effect on PMN chemotaxis was noted,suggesting SOD had no direct effect on PMNS, but prevented PMNrecruitment by decreasing the production of chemotactic mediators.Results indicate that acute lung damage from 48 h of hyperoxia andhyperventilation can be significantly ameliorated by a singleprophylactic dose of intratracheal SOD.

Materials and Methods Study Protocol

24 newborn piglets, 1.3±0.3 kg (1-2 days old)

Group I, positive control--10 hyperventilated for 48 hours (PaCO₂ 15-20torr), FiO₂ 1.0, (baseline, 24 h, 48 h studies).

Group II--8 treated as above, but given a single IT dose of recombinanthuman Cu/Zn SOD (5 mg/kg in saline).

Group III, negative control--6 normally ventilated for 48 hours (PaCO₂35-45 torr), FiO₂ 0.21.

Tracheal Aspirates

PMN chemotactic activity of Groups I, II and III (FIG. 1). Cell countsand differentials of Groups I, II and III (FIG. 2). Elastase (FIG. 3).Albumin (FIG. 3). Total protein concentration (FIG. 5).

Pulmonary Function Tests

Lung compliance (FIG. 4).

Bronchoalveolar Lavage

Quantitative and qualitative surfactant analysis: No negative effect onthe integrity of surfactant from administration of superoxide dismutasewas observed. Lung compliance improved with surfactant in lung.Furthermore, no negative effect of surfactant on the integrity ofsuperoxide dismutase was observed.

Pathologic Examination

Light microscopy as well as scanning and transmission electronmicroscopy was used to study the pathologic effects of oxygen andmechanical ventilation on the newborn piglets.

Conclusions

SOD facilitates the conversion of superoxide radicals (O₂ ⁻) to hydrogenperoxide (H₂ O₂). With fewer toxic oxygen radicals present, cell damageis minimized, chemotactic agents for PMNs are not produced, and acutelung injury is reduced.

Hyperoxia and hyperventilation for 48 hours causes significant cellular,biochemical and pathophysiologic changes in the lung. SOD moderated thedevelopment of acute lung injury as evidenced by decreased PMNinfiltration, elastase activity, protein influx and pathophysiologicabnormalities. Despite increases in protein influx, pulmonary surfactantremained active, and therefore SOD does not adversely affect theactivity of pulmonary surfactant.

References

1. Crapo et al., The Failure of Aerosolized Superoxide Dismutase toModify Pulmonary Oxygen Toxicity, American Review of Respiratory Disease115:1027-1033).

2. Seo, S.C. et al., The Effect of Intratracheally Administered Free andLiposome Entrapped Superoxide Dismutase and Catalase on ExperimentallyExposed Hyperoxic Injury in Rats' Lungs, Chung-Ang Journal of Medicine12(2):259-271 (June 1987).

3. Padmanabhan, R.V. et al., Protection Against Pulmonary OxygenToxicity in Rats by the Intratracheal Administration ofLiposome-Encapsulated Superoxide Dismutase or Catalase¹⁻³, Am. Rev.Respir. Dis. 132:164-167 (1985).

4. Turrens, J. F. et al., Protection Against Oxygen Toxicity byIntravenous Injection of Liposome-Entrapped Catalase and SuperoxideDismutase, J. Clin. Invest. 73:87-95 (Jan. 1984).

5. Crapo et al., Structural and Biochemical Changes in Rat LungsOccurring During Exposures to Lethal and Adaptive Doses of Oxygen,American Review of Respiratory Disease, 123:123-143 (1980).

6. Walther, F.J. et al., Prevention of Oxygen Toxicity with SuperoxideDismutase and Catalase in Premature Lambs, Journal of Free Radicals inBiology & Medicine 2:289-293 (1986).

7. A. M. Gonenne et al., JAP 67:1007-1042 (1989).

What is claimed is:
 1. A method of protecting a human from lung injurydue to hyperoxia and hyperventilation which comprises intratracheallyadministering to the human an amount of free CuZnSOD effective toprotect the human from lung injury due to hyperoxia andhyperventilation.
 2. A method of claim -, wherein the free CuZnSOD isadministered in the form of an aerosol.
 3. A method of claim wherein theintratracheal administration of free CuZnSOD is performed byinstillation.
 4. A method of claim 1, wherein the human is an adult. 5.A method of claim 1, wherein the human is a neonate.
 6. A method ofclaim 5, wherein the neonate is premature.
 7. A method of claim 6,wherein a surfactant is administered prior to administering the freeCuZnSOD.
 8. A method of claim 1, wherein the amount of CuZnSOD is fromabout 0.5 mg/kg to about 50 mg/kg of body weight of the human.
 9. Amethod of claim -, wherein the CuZnSOD is recombinant CuZnSOD.
 10. Amethod of claim 1, wherein the CuZnSOD is human CuZnSOD.